CN112618786A

CN112618786A - Ultraviolet cross-linking drug-loaded gel and preparation method thereof

Info

Publication number: CN112618786A
Application number: CN202011506341.8A
Authority: CN
Inventors: 魏健; 石艳丽; 邵萌; 王成山; 郭学平
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-04-09
Anticipated expiration: 2040-12-18
Also published as: CN112618786B

Abstract

The invention provides a preparation method of ultraviolet cross-linked drug-loaded gel, which comprises the following steps: dissolving methacryloylated hyaluronic acid or a salt thereof, zinc hyaluronate and a drug in water to obtain a first solution; adding a photoinitiator and a catalyst into the first solution, and filtering and sterilizing to obtain a second solution; and irradiating the second solution by ultraviolet light to perform a cross-linking reaction to obtain the drug-loaded gel. Compared with the prior art, the invention has the following technical effects: the ultraviolet cross-linked gel is subjected to filtration sterilization instead of damp-heat sterilization, so that the problem of degradation of the gel due to high temperature is effectively avoided; the ultraviolet cross-linked gel can load medicines sensitive to oxygen and high temperature, and the medicines can exert the maximum treatment effect due to the slow release effect of the cross-linked gel while the medicines are effectively protected; the ultraviolet cross-linked gel has good biocompatibility, and the low molecular weight zinc hyaluronate added in the gel can effectively inhibit the growth of bacteria and promote the healing of wounds, and has the synergistic effect with the loaded medicine.

Description

Ultraviolet cross-linking drug-loaded gel and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to ultraviolet cross-linked medicine-carrying gel and a preparation method thereof.

Background

Hyaluronic acid is a polysaccharide formed by connecting disaccharide units consisting of D-glucuronic acid and N-acetylglucosamine through glycosidic bonds, and widely exists in tissues such as placenta, amniotic fluid, crystalline lens, articular cartilage, skin dermis and the like; the hyaluronic acid has special physicochemical properties and biocompatibility, and is widely applied to wound repair, 3D printing, cosmetic filling and ophthalmic surgery viscoelastic agents.

Natural hyaluronic acid is sensitive to strong acids, strong bases, heat, free radicals and hyaluronidase, and is easily degraded, and modification thereof can overcome these disadvantages. The hyaluronic acid has a plurality of sites capable of being chemically modified, mainly comprises carboxyl, hydroxyl and the like, and the modified hyaluronic acid is endowed with new characteristics and exerts the value to the maximum extent. Currently, hyaluronic acid is modified by crosslinking modification. However, chemical crosslinking agents such as divinyl sulfone (DVS) and 1, 4-butanediol diglycidyl ether (BDDE) are mostly used as the crosslinking agents in the market, and the crosslinking agents are toxic or carcinogenic, which is not favorable for product safety, and the biocompatibility of the hyaluronic acid gel is correspondingly reduced with the increase of the dosage of the crosslinking agents.

Moreover, the hyaluronic acid gel modified by crosslinking has high viscosity, and cannot be sterilized by filtration, and currently, the sterility of the hyaluronic acid gel is mainly controlled by the following two ways: (1) the sterility of the product is ensured by process control and aseptic operation; this approach has high requirements on production processes and the environment and cannot guarantee complete sterility. (2) A terminal sterilization process is adopted to ensure the sterility of the product; the terminal sterilization method generally comprises moist heat sterilization, ethylene oxide sterilization, and,⁶⁰Co-gamma sterilization, cold ionizing radiation sterilization, and the like. Patent CN106074213A discloses a sodium hyaluronate gel for water light injection, and a preparation method and application thereof, wherein the sodium hyaluronate gel is subjected to terminal moist heat sterilization to obtain a sterile injection gel. However, these methods can cause irreversible changes in the polymer structure or affect the stability and activity of some drug-loaded gel drugs, and ultimately affect the product performance, while the gel is end-sterilized by chemical or physical means.

With the development of organic chemistry, techniques such as photocrosslinking and self-crosslinking have emerged. Patent CN107200854A discloses a preparation method of a hyaluronic acid hydrogel matrix for ultraviolet 3D printing, which is formed by synthesizing methacrylic acylated sodium hyaluronate with grafted alkenyl group and wool polypeptide nanospheres. The hydrogel matrix prepared by the preparation method makes full use of the characteristic of sulfydryl-alkene click reaction, and can be rapidly formed by gel under the irradiation of ultraviolet light, but the related description on the sterility guarantee of the gel is not provided.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method and application of ultraviolet drug-loaded gel.

Specifically, the present invention relates to the following aspects:

1. a preparation method of ultraviolet cross-linked drug-loaded gel is characterized by comprising the following steps:

dissolving methacryloylated hyaluronic acid or a salt thereof, zinc hyaluronate and a drug in water to obtain a first solution;

adding a photoinitiator and a catalyst into the first solution, and filtering and sterilizing to obtain a second solution;

and irradiating the second solution by ultraviolet light to perform a cross-linking reaction to obtain the drug-loaded gel.

2. The production method according to item 1, wherein the mass percentage of the methacrylated hyaluronic acid or salt thereof in the first solution is 0.1% to 2%, preferably 0.2% to 1.8%, and more preferably 0.5% to 1.5%, the mass percentage of the zinc hyaluronate is 0.01% to 2%, preferably 0.05% to 1.5%, and more preferably 0.2% to 1%, and the mass percentage of the drug is 0.01% to 0.3%, preferably 0.05% to 0.2%, and more preferably 0.1% to 0.15%.

3. The production method according to item 1 or 2, characterized in that the degree of substitution of the methacrylated hyaluronic acid or a salt thereof is 5% to 20%, preferably 7% to 15%, further preferably 9% to 13%, the molecular weight is 100-1000kDa, preferably 110-800kDa, further preferably 110-500kDa, and the molecular weight of the zinc hyaluronate is 50-1000kDa, preferably 100-700kDa, further preferably 200-500 kDa.

4. The process according to any one of claims 1 to 3, wherein the drug is one or more selected from the group consisting of vitamin C, vitamin A, vitamin D, aminopyrine, dipyrone, levofloxacin, ibuprofen, flufenamic acid, diclofenac sodium, phenylbutazone, gentamicin, tetracycline and chloramphenicol.

5. The preparation method according to any one of items 1 to 4, characterized in that the irradiation wavelength of the ultraviolet light is 200-400nm, preferably 254-365nm, and more preferably 365 nm.

6. The production method according to any one of claims 1 to 5, wherein the irradiation intensity of the ultraviolet irradiation is 2 to 100Mw/cm²Preferably 5 to 50Mw/cm²More preferably 15 to 30Mw/cm²。

7. The production method according to any one of claims 1 to 6, wherein the irradiation time of the ultraviolet light is 1 to 30min, preferably 5 to 15 min.

8. The production method according to any one of items 1 to 7, characterized in that the mass percentage of the photoinitiator in the second solution is 0.01% to 0.2%, the mass percentage of the catalyst is 0.03% to 0.3%, wherein the photoinitiator is selected from one or more of 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (Irgacure2959), 1-hydroxy-cyclohexylphenyl methanone (HCPK), phenylbis (2,4, 6-trimethylbenzoyl) phosphine oxide (Irgacure 819) and 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide (Irgacure TPO), the catalyst is selected from one or more of beta-hydroxyethyl methacrylate (HEMA), N-vinyl pyrrolidone (NVP) and styrene (St).

9. The method according to any one of claims 1 to 8, wherein the filtration sterilization is one or two selected from the group consisting of microfiltration membrane filtration sterilization and cartridge filtration sterilization.

10. The method according to any one of claims 1 to 9, wherein the first solution further comprises an osmotic pressure adjusting agent and a pH adjusting agent, wherein the osmotic pressure adjusting agent is one or more selected from the group consisting of sodium chloride, glucose and glycerol, and the pH adjusting agent is one or more selected from the group consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate.

11. An ultraviolet cross-linking drug-loaded gel, which is characterized in that the ultraviolet cross-linking drug-loaded gel is prepared by the preparation method of any one of items 1 to 10.

12. The ultraviolet cross-linked drug-loaded gel of claim 11, wherein the ultraviolet cross-linked drug-loaded gel is used for wound healing, diabetic foot, tissue bulking agent, or ophthalmic surgical viscoelastic.

The invention has the following technical effects:

(1) the ultraviolet cross-linked gel prepared by the invention is subjected to filtration sterilization instead of damp-heat sterilization, and the problem that the gel is degraded due to high temperature is effectively avoided.

(2) The ultraviolet cross-linked gel prepared by the invention can load the medicine sensitive to oxygen and high temperature, effectively protects the medicine, and simultaneously has the slow release effect so that the medicine can play the maximum treatment effect.

(3) The ultraviolet cross-linked gel has good biocompatibility, and the low molecular weight zinc hyaluronate added in the gel can effectively inhibit the growth of bacteria and promote the healing of wounds, and has the synergistic effect with the loaded medicine.

Drawings

FIG. 1 is a scanning electron micrograph of an ultraviolet crosslinked gel according to example 1 of the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

The invention provides a preparation method of ultraviolet cross-linked drug-loaded gel, which comprises the following steps:

Wherein the salt of the methacrylated hyaluronic acid is one or more of sodium salt, potassium salt, magnesium salt, calcium salt and zinc salt of the methacrylated hyaluronic acid.

Zinc hyaluronate (ZnHA) is a zinc salt of hyaluronic acid and is prepared by ion exchange of zinc ions with sodium ions on the carboxyl groups of sodium hyaluronate. The zinc ions have antibacterial and antiseptic capabilities, the antibacterial mechanism of ZnHA is mainly that the zinc ions are dissolved out to perform contact bacteriostasis, the contact bacteriostasis means that the zinc ions with positive charges are adsorbed on the surface of bacteria with negative charges due to the existence of free carboxyl by means of coulomb attraction to damage the cell walls of the bacteria, then the zinc ions further penetrate the damaged cell walls to replace the positions of cations on the surface of the cell membranes to damage the cell membranes, further cytoplasm outflows are caused, and finally the cells die to achieve the antibacterial purpose. Meanwhile, the surplus zinc ions can permeate into the cell through the cell membrane to play a role in inhibiting bacteria. After the bacteria are killed, zinc ions are dissociated, the sterilization activity is repeated, and the antibacterial effect is durable.

In a specific embodiment, the mass percentage of the methacrylated hyaluronic acid or salt thereof in the first solution is 0.1% to 2%, and for example, may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, preferably 0.2% to 1.8%, and more preferably 0.5% to 1.5%. The mass percentage of the zinc hyaluronate may be, for example, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, preferably 0.05% to 1.5%, and more preferably 0.2% to 1%. The mass percentage of the drug is 0.01 to 0.3%, for example, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, preferably 0.05% to 0.2%, and more preferably 0.1% to 0.15%.

In a specific embodiment, the degree of substitution of the methacrylated hyaluronic acid or salt thereof is 5% to 20%, and for example, may be 5%, 7%, 9%, 13%, 15%, 20%, preferably 7% to 15%, and more preferably 9% to 13%. Wherein the degree of substitution is the ratio of the number of moles of methacryloyl groups attached to the hydroxyl groups of the hyaluronic acid to the number of moles of the disaccharide units of the hyaluronic acid. Specific measurement methods can be found in the references Synthesis of methylated hyaluronic acid with a labeled degree of residence of Polymer 2007; 48: 1915-20. The molecular weight of the methacrylated hyaluronic acid or salt thereof is 100-minus 1000kDa, and may be, for example, 100kDa, 110kDa, 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, preferably 110-minus 800kDa, and more preferably 110-minus 500 kDa.

In a specific embodiment, the molecular weight of the zinc hyaluronate is 50-1000kDa, such as 50kDa, 100kDa, 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, preferably 100-700kDa, and more preferably 200-500 kDa.

In a specific embodiment, the drug is selected from one or more of vitamin C, vitamin a, vitamin D, aminopyrine, analgin, levofloxacin, ibuprofen, flufenamic acid, diclofenac sodium, phenylbutazone, gentamicin, tetracycline, and chloramphenicol.

In a specific embodiment, the irradiation wavelength of the ultraviolet light is 200-400nm, preferably 254-365nm, and more preferably 365 nm.

In a specific embodiment, the ultraviolet light has an irradiance of from 2 to 100Mw/cm²Preferably 5 to 50Mw/cm²More preferably 15 to 30Mw/cm². Here, the illumination intensity is a physical term referring to the luminous flux of the received visible light per unit area.

In a specific embodiment, the irradiation time of the ultraviolet light is 1-30min, preferably 5-15 min.

In a specific embodiment, the mass percent of the photoinitiator in the second solution is 0.01-0.2%, the mass percent of the catalyst is 0.03-0.3%, wherein the photoinitiator is selected from one or more of 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (Irgacure2959), 1-hydroxy-cyclohexylphenyl methanone (HCPK), phenylbis (2,4, 6-trimethylbenzoyl) phosphine oxide (Irgacure 819) and 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide (Irgacure TPO), the catalyst is selected from one or more of beta-hydroxyethyl methacrylate (HEMA), N-vinyl pyrrolidone (NVP) and styrene (St).

In a specific embodiment, the filter sterilization is selected from one or both of microfiltration and cartridge filter sterilization. Wherein the microporous membrane filtration sterilization is preferably the microporous membrane filtration sterilization with the pore diameter of 0.2 μm.

In a specific embodiment, the first solution further comprises an osmotic pressure regulator and a pH regulator, wherein the osmotic pressure regulator is selected from one or more of sodium chloride, glucose and glycerol, and the pH regulator is selected from one or two of disodium hydrogen phosphate and sodium dihydrogen phosphate, and preferably the pH of the first solution is adjusted to 7.3-7.4.

Further, the ultraviolet cross-linking drug-loaded gel can be prepared into a freeze-dried drug-loaded sponge or film through freeze-drying.

The invention also provides the ultraviolet crosslinking medicine-carrying gel prepared by the preparation method.

In a specific embodiment, the uv cross-linked drug loaded gel is used for wound healing, diabetic foot, tissue bulking or ophthalmic surgical viscoelastic.

The ultraviolet crosslinking medicine-carrying gel realizes the preparation of the medicine-carrying gel in an ultraviolet crosslinking mode, does not use a crosslinking agent, ensures the sterility of each link, is easy to achieve the operating conditions, saves the terminal sterilization process, ensures the performance of the gel, and provides convenience and possibility for adding unstable medicines into the gel. The prepared drug-loaded gel has good swelling performance, and the expansion coefficients are all more than 50; the drug-loaded gel has low enzymolysis rate, which shows that the gel has good enzymolysis resistance and long effect maintaining time in vivo; after the medicine-carrying gel leaching liquor is added into a cell culture solution, the cell survival rate is over 91 percent, which shows that the gel prepared by the invention has good safety, high biocompatibility and no cytotoxicity.

Examples

The experimental methods used in the following examples are all conventional methods, unless otherwise specified.

Sodium hyaluronate and zinc hyaluronate used in the following examples and comparative examples were produced by Huaxi Biotech Co., Ltd. Other materials, reagents, etc., are commercially available without specific reference.

Example 1

(1) Preparation of sodium methacryloylated hyaluronate

Weighing 20g of sodium hyaluronate (molecular weight of 150kDa), adding 1000mL of water, stirring for dissolving, sequentially adding 22mL of triethylamine, 80mL of glycidyl methacrylate and 20g of tetrabutylammonium chloride, stirring uniformly, reacting at 25 ℃ for 24 hours, precipitating with acetone, filtering, washing, collecting a filter cake after the reaction is finished, dissolving with water, repeating for 3 times, and freeze-drying to obtain 18.5g of sodium methacryloylated hyaluronate, wherein the molecular weight of the sodium methacryloylated hyaluronate is 110kDa, and the substitution degree of the sodium methacryloylated hyaluronate is 10.4%.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 0.5g of the prepared sodium methylacryloylated hyaluronate, adding 100mL of phosphate buffer solution with pH of 7.3, stirring until the sodium methylacryloylated hyaluronate is dissolved, then sequentially adding 1g of zinc hyaluronate (molecular weight of 500kDa) and 0.15g of diclofenac sodium, and stirring until the sodium methylacryloylated hyaluronate is dissolved uniformly. Adding photoinitiator I29590.01g and catalyst N-vinyl pyrrolidone 0.05g, stirring, filtering with 0.2 μm microporous membrane to obtain drug-carrying solution, transferring the drug-carrying solution into a glass culture dish, turning on 365nm strong ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to 22mw/cm²And irradiating by ultraviolet light for 15min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in a sterile environment to obtain the ultraviolet crosslinked drug-loaded gel.

(3) Scanning electron microscope observation of ultraviolet crosslinked gel

And (3) freeze-drying the prepared gel sample, spraying gold, observing by a scanning electron microscope, wherein the accelerating voltage is 5kV, and each sample is observed in 5-10 areas. The scanning electron micrograph of the UV crosslinked gel is shown in FIG. 1.

Example 2

(1) Preparation of sodium methacryloylated hyaluronate

Weighing 10g of sodium hyaluronate (molecular weight of 1100kDa), adding 1000mL of water, stirring for dissolving, sequentially adding 22mL of triethylamine, 22mL of glycidyl methacrylate and 10g of tetrabutylammonium chloride, stirring uniformly, reacting at 25 ℃ for 24 hours, precipitating with acetone, filtering, washing, collecting a filter cake after the reaction is finished, dissolving with water, repeating for 3 times, and freeze-drying to obtain 8.3g of sodium methacrylate acylate, wherein the molecular weight is 980kDa and the substitution degree is 5.2%.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 1.8g of the prepared sodium methacrylate acylated hyaluronate, adding 100mL of phosphate buffer solution with the pH value of 7.3, stirring until the sodium methacrylate acylated hyaluronate is dissolved, then sequentially adding 1.5g of zinc hyaluronate (the molecular weight is 100kDa) and 0.15g of diclofenac sodium, stirring until the zinc hyaluronate and the diclofenac sodium are dissolved uniformly, adding 29590.1 g of photoinitiator and 0.05g of catalyst N-vinyl pyrrolidone, stirring uniformly, passing through a 0.2 mu m microporous filter membrane to obtain a drug-loaded solution, transferring the drug-loaded solution into a glass culture dish, starting a 365nm strong ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to be 50mw/cm²And irradiating by ultraviolet light for 5min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in an aseptic environment to obtain the ultraviolet crosslinked drug-loaded gel.

Example 3

(1) Preparation of sodium methacryloylated hyaluronate

Weighing 10g of sodium hyaluronate (molecular weight of 850kDa), adding 1000mL of water, stirring for dissolving, sequentially adding 22mL of triethylamine, 60mL of glycidyl methacrylate and 10g of tetrabutylammonium chloride, stirring uniformly, reacting at 25 ℃ for 24 hours, precipitating with acetone, filtering, washing, collecting a filter cake after the reaction is finished, dissolving with water, repeating for 3 times, and freeze-drying to obtain 8.0g of sodium methacrylate acylate, wherein the molecular weight is 780kDa and the substitution degree is 14.1%.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 0.2g of the prepared sodium methylacryloyl hyaluronate, adding 100mL of phosphate buffer solution with pH of 7.3, stirring to dissolve, and sequentially adding0.05g of zinc hyaluronate (molecular weight of 700kDa) and 0.15g of diclofenac sodium, stirring until the zinc hyaluronate and the diclofenac sodium are uniformly dissolved, adding 0.1g of photoinitiator I29590.02g and catalyst N-vinyl pyrrolidone, stirring uniformly, filtering through a 0.2-micrometer microporous filter membrane to obtain a drug-carrying solution, transferring the drug-carrying solution into a glass culture dish, starting a 365nm strong ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to be 22mw/cm²And irradiating by ultraviolet light for 10min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in an aseptic environment to obtain the ultraviolet crosslinked drug-loaded gel.

Example 4

(1) Sodium methacryloylated hyaluronate was prepared as in example 1.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 0.1g of the prepared sodium methacrylate acylated hyaluronate, adding 100mL of phosphate buffer solution with the pH value of 7.3, stirring until the sodium methacrylate acylated hyaluronate is dissolved, then sequentially adding 2g of zinc hyaluronate (the molecular weight is 50kDa) and 0.15g of diclofenac sodium, stirring until the sodium hyaluronate is dissolved uniformly, adding 0.3g of photoinitiator I29590.2g and catalyst N-vinyl pyrrolidone, stirring uniformly, passing through a 0.2 mu m microporous filter membrane to obtain a drug-loaded solution, transferring the drug-loaded solution into a glass culture dish, starting a 365nm strong light ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to be 10mw/cm²And irradiating by ultraviolet light for 30min to obtain gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in an aseptic environment to obtain the ultraviolet crosslinked drug-loaded gel.

Example 5

(1) Sodium methacryloylated hyaluronate was prepared as in example 1.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 2.0g of the prepared sodium methacrylate acylated hyaluronate, adding 100mL of phosphate buffer solution with the pH value of 7.3, stirring until the sodium methacrylate acylated hyaluronate is dissolved, then sequentially adding 0.01g of zinc hyaluronate (the molecular weight is 1000kDa) and 0.15g of diclofenac sodium, stirring until the sodium hyaluronate is dissolved uniformly, adding 29590.01g of photoinitiator and 0.05g of catalyst N-vinyl pyrrolidone, stirring uniformly, and then passing throughObtaining a drug-carrying solution through a 0.2 mu m microporous filter membrane, transferring the drug-carrying solution into a glass culture dish, starting a 365nm strong light ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to be 22mw/cm²And irradiating by ultraviolet light for 15min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in a sterile environment to obtain the ultraviolet crosslinked drug-loaded gel.

Example 6

(1) Sodium methacryloylated hyaluronate was prepared as in example 1.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 1.5g of the prepared sodium methylacryloylated hyaluronate, adding 100mL of phosphate buffer solution with pH of 7.3, stirring until the sodium methylacryloylated hyaluronate is dissolved, then sequentially adding 0.2g of zinc hyaluronate (molecular weight of 200kDa) and 0.15g of diclofenac sodium, and stirring until the sodium methylacryloylated hyaluronate is dissolved uniformly. Adding photoinitiator I29590.01g and catalyst N-vinyl pyrrolidone 0.05g, stirring, filtering with 0.2 μm microporous membrane to obtain drug-carrying solution, transferring the drug-carrying solution into a glass culture dish, turning on 365nm strong ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to 22mw/cm²And irradiating by ultraviolet light for 15min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in a sterile environment to obtain the ultraviolet crosslinked drug-loaded gel.

Example 7

(1) Preparation of sodium methacryloylated hyaluronate

Weighing 10g of sodium hyaluronate (molecular weight of 1100kDa), adding 1000mL of water, stirring for dissolving, sequentially adding 22mL of triethylamine, 100mL of glycidyl methacrylate and 10g of tetrabutylammonium chloride, stirring uniformly, reacting at 25 ℃ for 24 hours, precipitating with acetone, filtering, washing, collecting a filter cake after the reaction is finished, dissolving with water, repeating for 3 times, and freeze-drying to obtain 9.5g of sodium methacryloylated hyaluronate, wherein the molecular weight of the sodium methacryloylated hyaluronate is 420kDa, and the substitution degree of the sodium methacryloylated hyaluronate is 25.2%.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

Weighing 1.5g of the prepared sodium methylacryloyl hyaluronate, and adding100mL of phosphate buffer solution with the pH value of 7.3 is stirred until the phosphate buffer solution is dissolved, then 0.2g of zinc hyaluronate (with the molecular weight of 200kDa) and 0.15g of diclofenac sodium are sequentially added, and the mixture is stirred until the phosphate buffer solution is dissolved uniformly. Adding photoinitiator I29590.01g and catalyst N-vinyl pyrrolidone 0.05g, stirring, filtering with 0.2 μm microporous membrane to obtain drug-carrying solution, transferring the drug-carrying solution into a glass culture dish, turning on 365nm strong ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to 22mw/cm²And irradiating by ultraviolet light for 15min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in a sterile environment to obtain the ultraviolet crosslinked drug-loaded gel.

Comparative example 1

Comparative example 1 is different from example 1 in that zinc hyaluronate having a molecular weight of 1200kDa is used in the preparation step of the ultraviolet cross-linked drug-loaded gel, and other reaction conditions are the same as those of example 1.

Comparative example 2

Comparative example 2 is different from example 1 in that zinc hyaluronate having a molecular weight of 30kDa is used in the preparation step of the ultraviolet cross-linked drug-loaded gel, and other reaction conditions are the same as those of example 1.

Comparative example 3

(1) Preparation of sodium methacryloylated hyaluronate

Weighing 10g of sodium hyaluronate (molecular weight of 1470kDa), adding 1000mL of water, stirring for dissolving, sequentially adding 22mL of triethylamine, 60mL of glycidyl methacrylate and 10g of tetrabutylammonium chloride, stirring uniformly, reacting at 25 ℃ for 24 hours, precipitating with acetone, filtering, washing, collecting a filter cake after the reaction is finished, dissolving with water, repeating for 3 times, and freeze-drying to obtain 8.7g of sodium methylacrylylated hyaluronate, wherein the molecular weight of 1280kDa and the substitution degree of 10.3%.

(2) Preparation of ultraviolet cross-linked drug-loaded gel

The procedure for preparation of the UV cross-linked drug loaded gel was the same as in example 1.

Comparative example 4

The procedure for the preparation of sodium methacryloylated hyaluronate was the same as in example 1.

0.5g of the prepared sodium methylacryloyl hyaluronate is weighed, 100mL of phosphate buffer solution with the pH value of 7.3 is added, the mixture is stirred until the mixture is dissolved, and then 0.15g of diclofenac sodium is added, and the mixture is stirred until the mixture is dissolved uniformly. Adding photoinitiator I29590.01g and catalyst N-vinyl pyrrolidone 0.05g, stirring, filtering with 0.2 μm microporous membrane to obtain drug-carrying solution, transferring the drug-carrying solution into a glass culture dish, turning on 365nm strong ultraviolet lamp, and adjusting the intensity of ultraviolet light reaching the glass culture dish to 22mw/cm²And irradiating by ultraviolet light for 15min to obtain a solution which is changed into gel, uniformly stirring and mixing the crosslinked gel and the non-crosslinked solution on the surface of the gel, and filling the mixture into a pre-sterilized container in a sterile environment to obtain the ultraviolet crosslinked drug-loaded gel.

The molecular weights and the amounts of addition of the respective materials of the above examples and comparative examples are shown in Table 1.

TABLE 1 molecular weight or addition amount of each of the materials of examples and comparative examples

Test example 1 gel swelling Properties

The mass (QM) -based hydrogel expansion coefficient was calculated by dividing the swollen gel mass (Ms) by the xerogel mass (Md).

The 11 groups of hydrogels were pre-soaked in Phosphate Buffered Saline (PBS) overnight, cut into small pieces (10-20mg), placed into the thermogravimetric analyzer weigh pans, respectively, and the initial swell was recorded as Ms; the sample was then slowly heated to 90-100 ℃ to reach a constant mass, and the final mass obtained was recorded as Md. The expansion coefficients of the hydrogels of group 11 are shown in Table 2.

TABLE 2 expansion coefficient of drug loaded gel

The gel swelling performance is an important index for evaluating the gel performance, the expansion coefficient is high, and the gel has strong capability of absorbing wound exudate when being used for wound dressings and the like.

The drug-loaded gel prepared in the embodiment of the invention has good swelling performance, the expansion coefficients are all more than 50, especially the expansion coefficient of the embodiment 1 is the highest, and the expansion coefficient is improved by more than 17.3 compared with the drug-loaded gel prepared in the comparative example.

Test example 2 enzymatic resistance

Precisely weighing 1g of 11 groups of gels, adding 1mL of 0.1mol/L phosphate buffer solution (pH7.0) and 500 mu L of hyaluronidase solution (600U/mL), uniformly mixing, placing in a water bath at 37 ℃, uniformly mixing 500 mu L of mixed solution and 1mL of 0.1mol/L phosphate buffer solution (pH7.0) in 6h, 12h, 24h and 48h, boiling to remove enzyme, centrifuging to obtain supernatant, detecting the light absorption value at 530nm according to a carbazole chromogenic method, and calculating the enzymolysis rate, wherein the enzymolysis rate is (the content of degraded cross-linked hyaluronic acid/the initial content of cross-linked hyaluronic acid) multiplied by 100%. The lower the enzymolysis rate, the better the gel is resistant to enzymolysis, and the longer the gel is in vivo. The results are shown in Table 3.

TABLE 3 enzymatic hydrolysis rate of drug loaded gels

The data in the table show that the drug-loaded gel prepared by the embodiment of the invention has good enzymolysis resistance, the enzymolysis rate in 48 hours is 59.8% or less, and the enzymolysis resistance of the embodiment 1 is optimal. In the comparative example, the molecular weight of zinc hyaluronate or the molecular weight and substitution degree of sodium methacrylate are not properly selected, and the enzymolysis resistance is reduced.

Test example 3 drug loaded gel biocompatibility test

1. Preparation of gel extract

According to the national standard of the people's republic of China, part 12 of the biological evaluation of medical instruments: sample preparation and reference samples. And (3) disinfecting the gel through an ultraviolet ozone box, putting the material into a sterile leaching container in a sterile ultra-clean workbench, completely soaking the disinfected material in the solution, and adopting a serum-free DMEM medium as a medium for leaching the material. Leaching for 48h, filtering and sterilizing the leaching liquor, sealing, and storing at 4 ℃ for later use.

2. Cell culture

L929 cells were selected and tested for in vitro cytotoxicity on UV cross-linked gels using the thiazole blue (MTT) method. The L929 cells were treated with 10⁴Perwell in 96-well plates, 5% CO₂Culturing at 37 deg.C for 24 hr; adding DMEM culture solution containing 5% of the 11 groups of gel leaching liquor into each well, adding DMEM culture solution into a blank control group, and continuously culturing for 24 hours to perform MTT test; measuring the light absorption value of the solution at 492nm by using a microplate reader, and calculating the cell survival rate (percent) A_{Sample (I)}/A_ControlX 100%. The results are shown in Table 4.

TABLE 4 cell viability results

	Cell survival rate
		Example 1	98.6％
Example 2	94.1％
		Example 3	94.8％
Example 4	92.0％
		Example 5	92.5％
Example 6	95.9％
		Example 7	93.0％
Comparative example 1	90.2％
		Comparative example 2	91.6％
Comparative example 3	91.9％
		Comparative example 4	91.3％

As can be seen from the table above, when the drug-loaded gel leaching liquor is added into the cell culture solution, the cell survival rate of the embodiment of the invention is more than 92%, which shows that the gel prepared by the invention has good safety, high biocompatibility and no cytotoxicity. In contrast, in the comparative example, the cell survival rate was reduced due to improper selection of the molecular weight and the addition amount of zinc hyaluronate and methacryloylated hyaluronic acid.

Test example 4 wound repair test

120 SD rats were anesthetized systemically, the backs were shaved and exposed with an electric razor, a copper cylinder mold with a radius of 1.2cm was placed in a water bath kettle at a constant temperature of 80 ℃ for 10min, and immediately placed on the backs of the rats for 8 seconds after being taken out, resulting in a burn model of the same degree with a radius of 1.2 cm. Randomly dividing 120 rats into 12 groups, treating the burn wound surfaces of the rats of 11 experimental groups by using the 11 groups of ultraviolet cross-linked hyaluronic acid gel, wherein the administration thickness is 3-5 mm, covering the wound surfaces by sterile gauze, then fixedly binding, spraying a commercially available diclofenac sodium spray on a control group, and treating the rest groups to be consistent with the experimental groups; the dressing is changed every other day until the crust skin completely falls off and the wound surface is covered by the epithelium. The dynamic change of wound healing was observed, the area of the crust surface was measured with a sterile transparent film for measurement at 1, 3, 5, 7, 10, 14d after burn, the wound healing rate was calculated, and the time to complete healing of the wound was recorded, the results are shown in table 5.

The wound healing rate is (eschar area on day 1-eschar area on day N)/eschar area on day 1 × 100%.

TABLE 5 burn wound healing rate and complete wound healing time in SD rats at different times

The test is terminated without healing of the wound

The drug-loaded gel prepared by the embodiment of the invention can well promote the healing of rat burn wound surfaces: after the drug-loaded gel prepared in the embodiment of the invention is applied to the wound surface of a rat for 14d, the wound surface of the rat burn reaches 100% of healing rate, and particularly, the complete healing of the wound surface of the rat burn is realized in the embodiments 1 and 10 d. Compared with the control group which uses the medicine without gel load, the wound healing rate and the complete healing time of the wound are both worse than the embodiment, which shows that the medicine-carrying gel of the invention effectively protects the medicine, and the cross-linked gel has the slow release effect which enables the medicine to play the maximum therapeutic effect.

Claims

2. The production method according to claim 1, wherein, in the first solution,

the mass percentage of the methacrylated hyaluronic acid or the salt thereof is 0.1% -2%, preferably 0.2% -1.8%, and more preferably 0.5% -1.5%;

the mass percentage of the zinc hyaluronate is 0.01-2%, preferably 0.05-1.5%, and more preferably 0.2-1%;

the mass percentage of the medicine is 0.01-0.3%, preferably 0.05-0.2%, and more preferably 0.1-0.15%.

3. The production method according to claim 1 or 2,

the degree of substitution of the methacryloylated hyaluronic acid or the salt thereof is 5% -20%, preferably 7% -15%, and more preferably 9% -13%, and the molecular weight is 100-800 kDa, preferably 110-800kDa, and more preferably 110-500 kDa;

the molecular weight of the zinc hyaluronate is 50-1000kDa, preferably 100-700kDa, and more preferably 200-500 kDa.

5. The method according to any one of claims 1 to 4, wherein the irradiation wavelength of the ultraviolet light is 200-400nm, preferably 254-365nm, and more preferably 365 nm.

6. The method according to any one of claims 1 to 5, wherein the ultraviolet irradiation is performed at an irradiation intensity of 2 to 100Mw/cm²Preferably 5 to 50Mw/cm²More preferably 15 to 30Mw/cm²。

7. The method according to any one of claims 1 to 6, wherein the irradiation time of the ultraviolet light is 1 to 30min, preferably 5 to 15 min.

8. The method according to any one of claims 1 to 7, wherein the photoinitiator is present in the second solution in an amount of 0.01 to 0.2% by mass and the catalyst is present in an amount of 0.03 to 0.3% by mass, wherein the photoinitiator is selected from one or more of 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (Irgacure2959), 1-hydroxy-cyclohexylphenyl ketone (HCPK), phenylbis (2,4, 6-trimethylbenzoyl) phosphine oxide (Irgacure 819) and 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide (Irgacure TPO), and the catalyst is selected from one or more of beta-hydroxyethyl methacrylate (HEMA), HEMA TPO, One or more of N-vinylpyrrolidone (NVP) and styrene (St).

10. The method according to any one of claims 1 to 9, wherein the first solution further comprises an osmotic pressure adjusting agent and a pH adjusting agent, wherein the osmotic pressure adjusting agent is one or more selected from the group consisting of sodium chloride, glucose and glycerol, and the pH adjusting agent is one or two selected from the group consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate.